Vlad C. Vasile

Phase I Trial of 17-Allylamino-17-Demethoxygeldanamycin in Patients With Advanced Cancer

PURPOSE We determined the maximum-tolerated dose (MTD) and the dose-limiting toxicities (DLT) of 17-allylamino-17-demethoxygeldanamycin (17-AAG) when infused on days 1, 8, and 15 of a 28-day cycle in advanced solid tumor patients. We also characterized the pharmacokinetics of 17-AAG, its effect on chaperone and client proteins, and whether cytochrome P450 (CYP) 3A5 and NAD(P)H:quinone oxidoreductase 1 (NQO1) polymorphisms affected 17-AAG disposition or toxicity. PATIENTS AND METHODS An accelerated titration design was used. Biomarkers were measured in peripheral-blood mononuclear cells (PBMCs) at baseline and on days 1 and 15, and pharmacokinetic analysis was performed on day 1 of cycle 1. CYP3A5*3 and NQO1*2 genotypes were determined and correlated with pharmacokinetics and toxicity. RESULTS Twenty-one patients received 52 courses at 11 dose levels. DLTs at 431 mg/m(2) were grade 3 bilirubin (n = 1), AST (n = 1), anemia (n = 1), nausea (n = 1), vomiting (n = 1), and myalgias (n = 1). No tumor responses were seen. 17-AAG consistently increased heat shock protein (Hsp) 70 levels in PBMCs. At the MTD, the clearance and half-life (t(1/2)) of 17-AAG were 11.6 L/h/m(2) and 4.15 hours, respectively; whereas the active metabolite 17-aminogeldanamycin had a t(1/2) of 7.63 hours. The CYP3A5*3 and NQO1*2 polymorphisms were not associated with 17-AAG toxicity. The CYP3A5*3 polymorphism was associated with higher 17-AAG clearance. CONCLUSION The MTD of weekly 17-AAG is 308 mg/m(2). 17-AAG induced Hsp70 in PBMCs, indicating that Hsp90 has been affected. Further evaluation of 17-AAG is ongoing using a twice-weekly regimen, and this schedule of 17-AAG is being tested in combination with chemotherapy.

Diseased skeletal muscle: a noncardiac source of increased circulating concentrations of cardiac troponin T.

OBJECTIVES The purpose of this study was to determine whether there is immunoreactive cardiac troponin T (cTnT) expression in diseased skeletal muscle that might cause possible false-positive increases in cTnT. BACKGROUND Cardiac troponin (I or T) is the biomarker of choice for the diagnosis of cardiac injury. Recently, we were presented with a case that challenged the specificity of cTnT. METHODS Patients with myopathies seen in the Neuromuscular Clinic at the Mayo Clinic were screened for increases in cTnT. If present, an assay for cTnI was performed. If normal, skeletal biopsy tissue was obtained for Western blot analysis using the capture and detection antibodies from both the fourth-generation and high-sensitivity cTnT assays. Results were compared with findings in normal cardiac tissue. RESULTS Sixteen patients had increases in cTnT but not cTnI. All had a myopathy by clinical evaluation, clinical testing, and biopsy. Four residual biopsy samples were obtained. All 3 antibodies used in the cTnT (M11.7, M7) and high-sensitivity cTnT (5D8, M7) assays were immunoreactive with a 37- to 39-kDa protein in all 4 diseased skeletal muscle biopsy specimens and in cardiac tissue. A second immunoreactive isoform (34 to 36 kDa) was also found in 1 patient. None of the noncardiac control tissues expressed immunoreactive protein. CONCLUSIONS These results document that there are forms in diseased skeletal muscle that could cause increases in circulating levels of cTnT. These increases could reflect re-expressed isoforms. Clinicians need to be aware of the possibility that noncardiac increases in cTnT may occur and lead to a possible false-positive diagnosis of cardiac injury when skeletal muscle pathology is present.

Biological and analytical variability of a novel high-sensitivity cardiac troponin T assay.

BACKGROUND High-sensitivity cardiac troponin assays will augment the frequency of increased results, making important the determination of reference change values to distinguish acute from chronic increases. We assessed short- and long-term biological variability of cardiac troponin T (cTnT) in healthy subjects with a novel high-sensitivity (hs) assay. METHODS We collected blood from 20 healthy volunteers at 5 time points for short-term study and biweekly at 4 times from the same volunteers for long-term study. We analyzed serum samples in duplicate with a hscTnT assay on the Roche Modular E170 and computed reference change values (RCVs) for analytical, intraindividual, interindividual, and total change values (CV(A), CV(I), CV(G), and CV(T), respectively) and the index of individuality (II). We calculated RCVs by using a log-normal approach, owing to the skewed results of the data. RESULTS Short- and long-term CV(A) values were 53.5% and 98%. CV(I) and CV(G) were 48.2% and 85.9%, respectively, for short-term studies and 94% and 94% for long-term studies. Mean delta values for the within-day study were 58% and -57.5%, and between-day mean delta values were 103.4% and -87%. Within- and between-day IIs were 0.8 and 0.14, respectively. CONCLUSIONS The biological variation demonstrated with the hscTnT assay is higher than prior data for cardiac troponin I. This may be attributed to differences in biology or assay imprecision at low concentrations. A short-term change (RCV log normal) of 85% and a long-term change of 315% is necessary to define a changing pattern.

Expedited PublicationDiseased Skeletal Muscle: A Noncardiac Source of Increased Circulating Concentrations of Cardiac Troponin T

OBJECTIVES The purpose of this study was to determine whether there is immunoreactive cardiac troponin T (cTnT) expression in diseased skeletal muscle that might cause possible false-positive increases in cTnT. BACKGROUND Cardiac troponin (I or T) is the biomarker of choice for the diagnosis of cardiac injury. Recently, we were presented with a case that challenged the specificity of cTnT. METHODS Patients with myopathies seen in the Neuromuscular Clinic at the Mayo Clinic were screened for increases in cTnT. If present, an assay for cTnI was performed. If normal, skeletal biopsy tissue was obtained for Western blot analysis using the capture and detection antibodies from both the fourth-generation and high-sensitivity cTnT assays. Results were compared with findings in normal cardiac tissue. RESULTS Sixteen patients had increases in cTnT but not cTnI. All had a myopathy by clinical evaluation, clinical testing, and biopsy. Four residual biopsy samples were obtained. All 3 antibodies used in the cTnT (M11.7, M7) and high-sensitivity cTnT (5D8, M7) assays were immunoreactive with a 37- to 39-kDa protein in all 4 diseased skeletal muscle biopsy specimens and in cardiac tissue. A second immunoreactive isoform (34 to 36 kDa) was also found in 1 patient. None of the noncardiac control tissues expressed immunoreactive protein. CONCLUSIONS These results document that there are forms in diseased skeletal muscle that could cause increases in circulating levels of cTnT. These increases could reflect re-expressed isoforms. Clinicians need to be aware of the possibility that noncardiac increases in cTnT may occur and lead to a possible false-positive diagnosis of cardiac injury when skeletal muscle pathology is present.

Elevated cardiac troponin is an independent risk factor for short- and long-term mortality in medical intensive care unit patients

Background:Troponin elevations are common in critically ill patients. Whether they are predictors of mortality independent of the severity of the underlying disease is unclear. Objective:To determine whether troponin elevations predict in-hospital, short-term, and long-term mortality in medical intensive care unit patients independent of the severity of the underlying disease as measured by Acute Physiology and Chronic Health Evaluation III prognostic system. Design:Retrospective study. Setting:We examined the Acute Physiology and Chronic Health Evaluation III database and cardiac troponin T levels of medical intensive care unit patients at Mayo Clinic, Rochester, MN. Patients:In all, 1,657 patients consecutively admitted to medical intensive care units between August 2000 and December 2001. Measurements:In-hospital, short-term (30-day), and long-term all-cause mortality. Results:During hospitalization, 12.5% of patients with a cardiac troponin T < 0.01 &mgr;g/L suffered deaths compared with 29.5% among those with cardiac troponin T ≥0.01 &mgr;g/L (p < .001). At 30 days, mortality was 13.7% without and 34.6% with elevations (p < .001). The expected probability of survival at 1-, 2-, and 3-yr follow-up was 43.7%, 33.8%, and 25.7% among patients with cardiac troponin T ≥0.01 &mgr;g/L and 75.3%, 67.6%, and 62.9% in those with cardiac troponin T < 0.01 &mgr;g/L, respectively (p < .001). After adjustment for the severity of disease and baseline characteristics, cardiac troponin levels were still associated with in-hospital, short-term, and long-term mortality (p = .006, p = .007, and p = .001, respectively). Limitations:This is a single-site retrospective study that included only patients in whom a troponin level was obtained on admission. Conclusions:In medical intensive care unit patients, admission troponin levels are independently associated with short- and long-term mortality, even after adjustment for severity of disease.

Genotoxic stress leads to centrosome amplification in breast cancer cell lines that have an inactive G1/S cell cycle checkpoint

Centrosome amplification plays a key role in the origin of chromosomal instability during cancer development and progression. In this study, breast cancer cell lines with different p53 backgrounds were used to investigate the relationship between genotoxic stress, G1/S cell cycle checkpoint integrity, and the development of centrosome amplification. Introduction of DNA damage in the MCF-7 cell line by treatment with hydroxyurea (HU) or daunorubicin (DR) resulted in the arrest of both G1/S cell cycle progression and centriole duplication. In these cells, which carry functional p53, HU treatment also led to nuclear accumulation of p53 and p21WAF1, retinoblastoma hypophosphorylation, and downregulation of cyclin A. MCF-7 cells carrying a recombinant dominant-negative p53 mutant (vMCF-7DNp53) exhibited a shortened G1 phase of the cell cycle and retained a normal centrosome phenotype. However, these cells developed amplified centrosomes following HU treatment. The MDA-MB 231 cell line, which carries mutant p53 at both alleles, showed amplified centrosomes at the outset, and developed a hyperamplified centrosome phenotype following HU treatment. In cells carrying defective p53, the development of centrosome amplification also occurred following treatment with another DNA damaging agent, DR. Taken together, these findings demonstrate that loss of p53 function alone is not sufficient to drive centrosome amplification, but plays a critical role in this process following DNA damage through abrogation of the G1/S cell cycle checkpoint. Furthermore, these studies have important clinical implications because they suggest that breast cancers with compromised p53 function may develop centrosome amplification and consequent chromosomal instability following treatment with genotoxic anticancer drugs.

Long-term prognostic significance of elevated cardiac troponin levels in critically ill patients with acute gastrointestinal bleeding.

Background:Elevations in troponin level have prognostic importance in critically ill patients, including those with gastrointestinal (GI) bleeding. However, there are no data addressing the independent association of troponin levels and mortality, adjusted for the severity of the underlying disease, in patients with GI bleeding. Objective:This study was designed to determine whether troponin T elevations are independently associated with in-hospital, short-term (30 days), and long-term mortality in medical intensive care unit patients with GI bleeding after adjusting for the severity of disease measured by the Acute Physiology, Age, and Chronic Health Evaluation score prognostic system. Design:Retrospective study. Setting:We examined the Acute Physiology, Age, and Chronic Health Evaluation III database and cardiac troponin T levels from patients consecutively admitted to the medical intensive care unit at Mayo Clinic, Rochester, MN, with acute GI bleeding. Patients:Between August 2000 and July 2005, 1076 patients with acute GI bleeding consecutively admitted to the medical intensive care units. Measurements:In-hospital, short-term (30 days), and long-term all-cause mortality. Results:During hospitalization, 8.0% of deaths occurred in patients with troponin T <0.01% and 11.9% with troponin T ≥0.01 (p = 0.083). At 30 days, mortality was 10.1% and 18.8% in patients without and with elevations of troponins, respectively (p < 0.001). The Kaplan-Meier expected probability of survival at 1-, 2-, and 3-yr follow-up was 54.2%, 40.8%, and 30.4% with troponin T ≥0.01 &mgr;g/L and 78.3%, 69.3%, and 61.5% with troponin T <0.01 &mgr;g/L (p < 0.001). After adjustment for severity of disease and baseline characteristics, cardiac troponin levels were associated only with long-term mortality (p < 0.001). Limitations:This is a retrospective, single-center study which included only patients in whom troponin level was determined upon admission. Conclusions:In patients with GI bleeding severe enough to require admission to the medical intensive care unit, admission troponin T elevations are associated with long-term but not short-term mortality.

TGFβ Inducible Early Gene-1 (TIEG1) and Cardiac Hypertrophy: Discovery and Characterization of a Novel Signaling Pathway

Cellular mechanisms causing cardiac hypertrophy are currently under intense investigation. We report a novel finding in the TGFβ inducible early gene (TIEG) null mouse implicating TIEG1 in cardiac hypertrophy. The TIEG−/− knock‐out mouse was studied. Male mice age 4–16 months were characterized (N = 86 total) using echocardiography, transcript profiling by gene microarray, and immunohistochemistry localized upregulated genes for determination of cellular mechanism. The female mice (N = 40) did not develop hypertrophy or fibrosis. The TIEG−/− knock‐out mouse developed features of cardiac hypertrophy including asymmetric septal hypertrophy, an increase in ventricular size at age 16 months, an increase (214%) in mouse heart/weight body weight ratio TIEG−/−, and an increase in wall thickness in TIEG−/− mice of (1.85 ± 0.21 mm), compared to the control (1.13 ± 0.15 mm, P < 0.04). Masson Trichrome staining demonstrated evidence of myocyte disarray and myofibroblast fibrosis. Microarray analysis of the left ventricles demonstrated that TIEG−/− heart tissues expressed a 13.81‐fold increase in pituitary tumor‐transforming gene‐1 (Pttg1). An increase in Pttg1 and histone H3 protein levels were confirmed in the TIEG−/− mice hearts tissues. We present evidence implicating TIEG and possibly its target gene, Pttg1, in the development of cardiac hypertrophy in the TIEG null mouse. J. Cell. Biochem. 100: 315–325, 2007.

Elevated Cardiac Troponin T Levels in Critically Ill Patients with Sepsis

BACKGROUND It is known that troponin elevations have prognostic importance in critically ill patients. We examined whether cardiac troponin T elevations are independently associated with in-hospital, short-term (30 days), and long-term (3 years) mortality in intensive care unit (ICU) patients admitted with sepsis, severe sepsis, and septic shock after adjusting for the severity of disease with the Acute Physiology, Age and Chronic Health Evaluation III system. METHODS We studied the Mayo Clinics Acute Physiology, Age and Chronic Health Evaluation III database and cardiac troponin T levels from patients admitted consecutively to the medical ICU. Between January 2001 and December 2006, 926 patients with sepsis had cardiac troponin T measured at ICU admission. In-hospital, short-term, and long-term all-cause mortality were determined. RESULTS Among study patients, 645 (69.7%) had elevated cardiac troponin T levels and 281 (30.3%) had undetectable cardiac troponin T. During hospitalization, 15% of the patients with troponin T <0.01 ng/mL died compared with 31.9% of those with troponin T ≥ 0.01 ng/mL (P < .0001). At 30 days, mortality was 31% and 17% in patients with and without elevations, respectively (P < .0001). The Kaplan-Meier probability of survival at 1-, 2-, and 3-year follow-ups was 68.1%, 56.3%, and 46.8% with troponin T ≥ 0.01 ng/mL, respectively, and 76.4%, 69.1%, and 62.0% with troponin T <0.01 μg/L, respectively (P < .0001). After adjustment for severity of disease and baseline characteristics, cardiac troponin T levels remained associated with in-hospital and short-term mortality but not with long-term mortality. CONCLUSIONS In patients with sepsis who are admitted to an ICU, cardiac troponin T elevations are independently associated with in-hospital and short-term mortality but not long-term mortality.

Significance of Elevated Cardiac Troponin T Levels in Critically Ill Patients with Acute Respiratory Disease

BACKGROUND Elevations in cardiac troponin have prognostic importance in critically ill patients. However, there are no data addressing the independent association between troponin levels and mortality, adjusted for the severity of the underlying disease, in patients hospitalized for acute respiratory disorders. We investigated whether troponin T (cTnT) elevations are independently associated with in-hospital mortality in patients in the intensive care unit (ICU) admitted for severe and acute respiratory conditions. After adjusting for the severity of disease measured by the Acute Physiology, Age, and Chronic Health Evaluation (APACHE) III prognostic system, we evaluated short-term (30 days) and long-term (3 years) mortality. METHODS We studied the APACHE III database and cTnT levels from patients admitted consecutively to the ICU at Mayo Clinic, Rochester, Minnesota. Between January 2001 and December 2005, 2078 patients with respiratory conditions had cTnT measured at ICU admission. In-hospital, short-term (30 days) and long-term (3 years) all-cause mortality were determined. RESULTS Of the study patients, 878 (42.3%) had elevated cTnT and 1200 patients (57.7%) had undetectable cTnT. During hospitalization, 1.1% of the patients with troponin T <0.01 ng/mL died compared to 21% of those with troponin T ≥0.01 ng/mL (P <.0001). At 30 days, mortality was 18.6% in patients with elevations of cTnT and 1.5% in patients without elevations of cTnT (P <.0001). The Kaplan-Meier probability of survival at 1-year follow-up was 71.0%, at 2-year follow-up was 48.3%, and 3-year follow-up was 39.4% with troponin T ≥0.01 ng/mL and at 1-year follow-up was 98.8%, at 2-year follow-up was 97.2%, and at 3-year follow-up was 95.5% with troponin T <0.01 μg/L (P <.0001). After adjustment for severity of disease and baseline characteristics, cTnT levels remained associated with in-hospital, short-term and long-term mortality (P <.0001). CONCLUSIONS In patients admitted to the ICU for respiratory disorders, cTnT elevations are independently associated with in-hospital, short-term and long-term mortality.